Process for selectively extracting procyanidins

ABSTRACT

A process for selectively extracting cocoa procyanidins from an aqueous mixture of cocoa polyphenols, by using a particular sequence of acetate solvents and/or diethyl ether solvents to extract selected desired procyanidin monomers and/or oligomers. The aqueous mixture of cocoa polyphenols is first extracted with n-butyl acetate. The acetate solvents include n-butyl acetate, ethyl acetate and methyl acetate. The diethyl ether solvents include mixtures of methyl acetate and diethyl ether in the following ratios (methyl acetate:diethyl ether): 0:100, 25:75, 75:25 (all v/v).

CROSS-REFERENCE TO RELATED APPLICATIONS

None.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a process for selectivelyextracting cocoa procyanidins according to their degree ofpolymerization, using particular sequences of solvents. In particular,the present invention is directed to a process for selectivelyextracting individual oligomers or small groups of oligomers from amixture of cocoa procyanidins, using n-butyl acetate as the firstextraction solvent.

It is known that individual flavan-3-ols, such as catechin, epicatechinand cocoa procyanidin oligomers, exhibit distinct properties and havedistinct applications for human and animal use. Selectively extractingspecific monomers or oligomers on the basis of degree of polymerizationwill allow for more targeted and efficacious use thereof, inpharmacological applications, as well as in food-grade products.

2. Discussion of the Related Art

Proanthocyanidins, the oligomers and polymers of flavan-3-ols, are thesecond most abundant natural plant phenol after lignin. The flavan-3-olsubunits are linked primarily through a carbon-carbon bond from the 4position of one subunit to the 8 position of another subunit (C4→C8),and to a lesser extent through C4→C6 linkage.

Procyanidins represent the largest class of proanthocyanidins. Gu et al.showed that out of 41 foods found to contain proanthocyanidins, 27contained procyanidins. (J. Agric. and Food Chem. 51 (2003) 7513).Procyanidins typically consist of (−)-epicatechin, (+)-epicatechin,(−)-catechin and/or (+)-catechin subunits.

Procyanidins include B-type and A-type proanthocyanidins. In B-typeproanthocyanidins, the monomeric subunits (catechin, epicatechin) areconnected via interflavan linkages of C4→C6 and/or C4→C8. Oligomers withexclusively C4→C8 linkages are linear, while the presence of at leastone C4→C6 bond results in a branched oligomer. By contrast, A-typeproanthocyanidins are doubly-linked oligomers, containing linkagesC2-O-C7 and C4→C6 or C4→C8.

The molecular weight of proanthocyanidins typically is expressed asdegree of polymerization (DP), and individual oligomers are commonlyreferred to as dimers, trimers, etc.

It is known that individual procyanidin oligomers present specificcharacteristics and potential benefits for use in humans and animals.For example, Tempesta discloses that procyanidin oligomers having adegree of polymerization (DP) of 2-11 possess significant antiviralactivity, and are useful in treating warm-blooded animals, includinghumans, infected with paramyxovaridae such as respiratory syncytialvirus, orthomyxovaridae such as influenza A, B and C, and herpes virusessuch as Herpes Simplex virus. (U.S. Pat. No. 5,211,944). Romanczyk Jr.,et al. disclose antineoplastic compositions comprising procyanidinoligomers having a DP of 3-11 together with a suitable carrier. (U.S.Pat. No. 5,554,645). Romanczyk, Jr. et al. also disclose thatprocyanidin oligomers having a DP of 5-12 are useful as antioxidants.(U.S. Pat. No. 5,891,905). Schmitz et al. disclose the use of cocoaprocyanidin oligomers (DP of 2-18) together with acetylsalicylic acid asanti-platelet therapy. (U.S. Pat. No. 6,524,630).

Significant improvements in the separation and resolution of procyanidinoligomers have been achieved. (See, Rigaud et al., Chromatogr. 654(1993) 179; Cheynier et al., Methods in Enzymology 299 (1999); Natsumeet al., Biosci. Biotechnol. Biochem. 64 (2000) 2581). Resolution ofprocyanidin oligomers up to the pentamer (DP=5) has been obtained.Hammerstone et al. disclosed modifications of this method, leading toimprovements in resolution of monomers through the nonamers in theanalysis of unfermented cacao seeds. (J. Agric. and Food Chem. 47 (1999)490). Gu et al. disclosed still further improvements, leading to theelution of a polymer peak (DP>10), as well as enhancement in overallpeak shape and resolution. (J. Agric. and Food Chem. 50 (2002) 4852).

It would be desirable to be able to maximize the loading capacity ofcocoa procyanidins onto a preparative scale HPLC system, so that greaterquantities of desired specific monomers and/or oligomers could be elutedfor further study or use. Theoretically, the loading capacity of apreparative scale HPLC column (300×55 mm, 100 μm) is approximately 4 gof material. However, cocoa procyanidin extracts present solubilityproblems that limit the maximum loading capacity to approximately 400mg, of which only approximately 41% are flavonoids.

It is expected that a significant increase in sample loading (flavanol)content can be achieved via selective extraction of individual or smallgroups of oligomers from a polyphenol mixture, prior to insertion ontoan HPLC column. Accordingly, what is needed is a method for selectivelyextracting other desired cocoa procyanidin oligomers from an aqueousextract of cocoa polyphenols on the basis of degree of polymerization.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a process for selectively extractingcocoa procyanidins from an aqueous mixture of cocoa polyphenols, byextracting the aqueous mixture with n-butyl acetate and separating anaqueous phase and an n-butyl acetate phase. The aqueous phase isenriched in procyanidin dimers and higher oligomers and the n-butylacetate phase is enriched in catechin and epicatechin.

In a preferred embodiment, the aqueous phase enriched in procyanidindimers and higher oligomers is extracted with an organic solvent whichis either diethyl ether or a mixture of methyl acetate and diethyl ethereffective to selectively extract catechin, epicatechin and procyanidindimers, and the phases are separated. The aqueous phase is enriched inprocyanidin trimers and higher oligomers and the organic phase isenriched in procyanidin dimers. Preferably, the mixture of methylacetate and diethyl ether is approximately 25:75 (v/v). Further in thisembodiment, the aqueous phase enriched in procyanidin trimers and higheroligomers is extracted with ethyl acetate and the phases are separated.The aqueous phase is enriched in procyanidin pentamers and higheroligomers and the ethyl acetate phase is enriched in procyanidin trimersand tetramers. Still further in this embodiment, the aqueous phaseenriched in procyanidin pentamers and higher oligomers is extracted withan organic solvent which is a mixture of methyl acetate and diethylether effective to selectively extract catechin, epicatechin andprocyanidin dimers, trimers, tetramers and pentamers, and the phases areseparated. The aqueous phase is enriched in procyanidin hexamers andhigher oligomers and the organic phase is enriched in procyanidinpentamers. Preferably, the mixture of methyl acetate and diethyl etheris approximately 75:25 (v/v). Still further in this embodiment, theaqueous phase enriched in procyanidin hexamers and higher oligomers isextracted with methyl acetate and the phases are separated. The aqueousphase is enriched in procyanidin nonamers and higher oligomers and themethyl acetate phase is enriched in procyanidin hexamers, heptamers andoctamers. Still further in this embodiment, the aqueous phase enrichedin procyanidin nonamers and higher oligomers is extracted with methylacetate and the phases are separated. The methyl acetate phase isenriched in procyanidin nonamers and higher oligomers.

In another embodiment, the aqueous phase enriched in procyanidin trimersand higher oligomers is extracted with methyl acetate and the phases areseparated. The aqueous phase is enriched in procyanidin nonamers andhigher oligomers and the methyl acetate phase is enriched in procyanidintrimers, tetramers, pentamers, hexamers, heptamers and octamers. Furtherin this embodiment, the solvent is removed from the methyl acetatephase, the resulting product is dissolved in water, and the dissolvedproduct is extracted with ethyl acetate. The phases are separated, withthe aqueous phase being enriched in procyanidin pentamers, hexamers,heptamers and octamers and the ethyl acetate phase being enriched inprocyanidin trimers and tetramers. Further in this embodiment, theaqueous phase enriched in procyanidin pentamers, hexamers, heptamers andoctamers is extracted with an organic solvent which is a mixture ofmethyl acetate and diethyl ether effective to selectively extractcatechin, epicatechin and procyanidin dimers, trimers, tetramers andpentamers and the phases are separated. The aqueous phase is enriched inprocyanidin hexamers, heptamers and octamers and the organic phase isenriched in procyanidin pentamers. Preferably, the mixture of methylacetate and diethyl ether is approximately 75:25 (v/v). Also in thisembodiment, the aqueous phase enriched in procyanidin nonamers andhigher oligomers is extracted with methyl acetate and the phases areseparated. The methyl acetate phase is enriched in procyanidin nonamersand higher oligomers.

In another embodiment, the aqueous phase enriched in procyanidin dimersand higher oligomers is extracted with ethyl acetate and the phases areseparated. The aqueous phase is enriched in procyanidin pentamers andhigher oligomers and the ethyl acetate phase is enriched in procyanidindimers, trimers and tetramers. In this embodiment, the aqueous phaseenriched in procyanidin pentamers and higher oligomers is extracted withan organic solvent which is a mixture of methyl acetate and diethylether effective to selectively extract catechin, epicatechin andprocyanidin dimers, trimers, tetramers and pentamers and the phases areseparated. The aqueous phase is enriched in procyanidin hexamers andhigher oligomers and the organic phase is enriched in procyanidinpentamers. Preferably, the mixture of methyl acetate and diethyl etheris approximately 75:25 (v/v). Further in this embodiment, the aqueousphase enriched in procyanidin hexamers and higher oligomers is extractedwith methyl acetate and the phases are separated. The aqueous phase isenriched in procyanidin nonamers and higher oligomers and wherein themethyl acetate phase is enriched in procyanidin hexamers, heptamers, andoctamers. Still further in this embodiment, the aqueous phase enrichedin procyanidin nonamers and higher oligomers is extracted with methylacetate and the phases are separated. The methyl acetate phase isenriched in procyanidin nonamers and higher oligomers. Also in thisembodiment, the solvent is removed from the ethyl acetate phase, theresulting product is dissolved in water, and the dissolved product isextracted in an organic solvent which is either diethyl ether or amixture of methyl acetate and diethyl ether effective to selectivelyextract catechin, epicatechin and procyanidin dimers. The phases areseparated. The aqueous phase is enriched in procyanidin trimers andtetramers and the organic phase is enriched in procyanidin dimers.Preferably, the mixture of methyl acetate and diethyl ether isapproximately 25:75 (v/v). Alternatively in this embodiment, the aqueousphase enriched in procyanidin pentamers and higher oligomers isextracted with methyl acetate and the phases are separated. The aqueousphase is enriched in procyanidin nonamers and higher oligomers and themethyl acetate phase is enriched in procyanidin pentamers, hexamers,heptamers and octamers. Also in this alternative, the solvent is removedfrom the methyl acetate phase, the resulting product is dissolved inwater, and the dissolved product is extracted with an organic solventwhich is a mixture of methyl acetate and diethyl ether effective toselectively extract catechin, epicatechin and procyanidin dimers,trimers, tetramers and pentamers. The phases are separated The aqueousphase is enriched in procyanidin hexamers, heptamers and octamers andthe organic phase is enriched in procyanidin pentamers. Preferably, themixture of methyl acetate and diethyl ether is approximately 75:25(v/v).

In another embodiment, the aqueous phase enriched in procyanidin dimersand higher oligomers is extracted with an organic solvent which is amixture of methyl acetate and diethyl ether effective to selectivelyextract catechin, epicatechin and procyanidin dimers, trimers, tetramersand pentamers, and the phases are separated. The aqueous phase isenriched in procyanidin hexamers and higher oligomers and the organicphase is enriched in procyanidin dimers, trimers, tetramers andpentamers. Preferably, the mixture of methyl acetate and diethyl etheris approximately 75:25 (v/v). In this embodiment, the aqueous phaseenriched in procyanidin hexamers and higher oligomers is extracted withmethyl acetate and the phases are separated. The aqueous phase isenriched in procyanidin nonamers and higher oligomers and the methylacetate phase is enriched in procyanidin hexamers, heptamers andoctamers. Further in this embodiment, the aqueous phase enriched inprocyanidin nonamers and higher oligomers is extracted with methylacetate and the phases are separated. The methyl acetate phase isenriched in procyanidin nonamers and higher oligomers. Alternatively inthis embodiment, the solvent is removed from the organic phase enrichedin procyanidin dimers, trimers, tetramers and pentamers, the resultingproduct is dissolved in water, and the dissolved product is extractedwith an organic solvent which is either diethyl ether or a mixture ofmethyl acetate and diethyl ether effective to selectively extractcatechin, epicatechin and procyanidin dimers. The phases are separated.The aqueous phase being is in procyanidin trimers, tetramers andpentamers and the organic phase is enriched in procyanidin dimers.Preferably, the mixture of methyl acetate and diethyl ether isapproximately 25:75 (v/v). Also in this alternative, the aqueous phaseenriched in procyanidin trimers, tetramers and pentamers is extractedwith ethyl acetate and the phases are separated. The aqueous phase isenriched in procyanidin pentamers and the ethyl acetate phase isenriched in procyanidin trimers and tetramers.

In a further embodiment, the aqueous phase enriched in procyanidindimers and higher oligomers is extracted with methyl acetate and thephases are separated. The aqueous phase is enriched in procyanidinnonamers and higher oligomers and the methyl acetate phase is enrichedin procyanidin dimers, trimers, tetramers, pentamers, hexamers,heptamers and octamers. In this embodiment, the solvent is removed fromthe methyl acetate phase, a product obtained thereby is dissolved inwater, and the dissolved product is extracted with an organic solventwhich is either diethyl ether or a mixture of methyl acetate and diethylether effective to selectively extract catechin, epicatechin andprocyanidin dimers. The phases are separated. The aqueous phase isenriched in procyanidin trimers, tetramers, pentamers, hexamers,heptamers and octamers and the organic phase is enriched in procyanidindimers. Preferably, the mixture of methyl acetate and diethyl ether isapproximately 25:75 (v/v). Further in this embodiment, the aqueous phaseenriched in procyanidin trimers, tetramers, pentamers, hexamers,heptamers, octamers and minor amounts of nonamers is extracted withethyl acetate and the phases are separated. The aqueous phase isenriched in procyanidin pentamers, hexamers, heptamers, octamers andminor amounts of procyanidin nonamers and the ethyl acetate phase isenriched in procyanidin trimers and tetramers. Still further in thisembodiment, the aqueous phase enriched in procyanidin pentamers,hexamers, heptamers, octamers and minor amounts of procyanidin nonamersis extracted with an organic solvent which is a mixture of methylacetate and diethyl ether effective to selectively extract catechin,epicatechin and procyanidin dimers, trimers, tetramers and pentamers,and the phases are separated. The aqueous phase is enriched inprocyanidin hexamers, heptamers, and octamers and the organic phase isenriched in procyanidin pentamers. Preferably, the mixture of methylacetate and diethyl ether is approximately 75:25 (v/v). Alternatively inthis embodiment, the solvent is removed from the methyl acetate phaseenriched in procyanidin dimers, trimers, tetramers, pentamers, hexamers,heptamers and octamers, the resulting product is dissolved in water, thedissolved product is extracted with an organic solvent which is amixture of methyl acetate and diethyl ether effective to selectivelyextract catechin, epicatechin and procyanidin dimers, trimers tetramersand pentamers. The phases are separated. The aqueous phase is enrichedin procyanidin hexamers, heptamers and octamers and the organic phase isenriched in procyanidin dimers, trimers, tetramers and pentamers.Preferably, the mixture of methyl acetate and diethyl ether isapproximately 75:25 (v/v). Also in this alternative, the solvent isremoved from the organic phase enriched in procyanidin dimers, trimers,tetramers and pentamers, the resulting product is dissolved in water,and the dissolved product is extracted with an organic solvent which iseither diethyl ether or a mixture of methyl acetate and diethyl ethereffective to selectively extract catechin, epicatechin and procyanidindimers. The phases are separated. The aqueous phase is enriched inprocyanidin trimers, tetramers and pentamers and the organic phase isenriched in procyanidin dimers. Preferably, the mixture of methylacetate and diethyl ether is approximately 25:75 (v/v). Also in thisalternative, the aqueous phase enriched in procyanidin trimers,tetramers and pentamers is extracted with ethyl acetate and the phasesare separated. The aqueous phase is enriched in procyanidin pentamersand the ethyl acetate phase is enriched in procyanidin trimers andtetramers.

These and other objects and embodiments are disclosed or will be obviousfrom the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: HPLC chromatogram of cocoa polyphenol standard.

FIG. 2: HPLC/FLD chromatograms of methyl acetate organic extractfraction from aqueous cocoa polyphenol extract.

FIG. 3: HPLC/FLD chromatogram of n-butyl acetate organic extractfraction from aqueous cocoa polyphenol extract.

FIG. 4: HPLC/FLD chromatogram of ethyl acetate organic extract fractionfrom aqueous cocoa polyphenol extract.

FIG. 5: HPLC/FLD chromatogram of diethyl ether organic extract fractionfrom aqueous cocoa polyphenol extract.

FIG. 6: HPLC/FLD chromatogram of organic extract fraction from aqueouscocoa polyphenol extract; solvent is a mixture of methyl acetate anddiethyl ether (25:75 v/v).

FIG. 7: HPLC/FLD chromatogram of organic extract fraction from aqueouscocoa polyphenol extract; solvent is a mixture of methyl acetate anddiethyl ether (50:50 v/v).

FIG. 8: HPLC/FLD chromatogram of organic extract fraction from aqueouscocoa polyphenol extract; solvent is a mixture of methyl acetate anddiethyl ether (75:25 v/v).

FIG. 9: Flow diagram of selective extraction pathways for selectiveextraction of an aqueous cocoa polyphenol extract, where the firstsolvent is n-butyl acetate.

FIG. 10: Flow diagram of selective extraction pathways for selectiveextraction of an aqueous cocoa polyphenol extract where the firstsolvent is ethyl acetate.

FIG. 11: Flow diagram of selective extraction pathways for selectiveextraction of an aqueous cocoa polyphenol extract where the firstsolvent is methyl acetate.

FIG. 12: Flow diagram of selective extraction pathways for selectiveextraction of an aqueous cocoa polyphenol extract where the firstsolvent is diethyl ether.

FIG. 13: Flow diagram of selective extraction pathways for selectiveextraction of an aqueous cocoa polyphenol extract where the firstsolvent is a mixture of methyl acetate and diethyl ether (25:75 v/v).

FIG. 14: Flow diagram of selective extraction pathways for selectiveextraction of an aqueous cocoa polyphenol extract where the firstsolvent is a mixture of methyl acetate and diethyl ether (75:25 v/v).

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses processes for selectively extractingindividual or small groups of cocoa procyanidin monomers and/oroligomers from an aqueous mixture of cocoa polyphenols. In particular,the invention is directed to selective extraction of procyanidinmonomers and/or oligomers via a selected sequence of acetate- and/ordiethyl ether-based solvents, wherein the first extraction solvent isn-butyl acetate.

It has been shown that dissolving a cocoa polyphenol extract in waterand extracting the aqueous solution with ethyl acetate will selectivelyextract the lower mass procyanidin oligomers (catechin, epicatechin,procyanidin dimers, trimers and tetramers). Based on these results,additional solvents and sequences of solvents that were immiscible inwater and yet polar, “green,” food grade, and reasonably priced, havebeen examined. Specifically, selective extraction of cocoa procyanidinmonomers and/or oligomers with n-butyl acetate and methyl acetate hasbeen examined, both alone and in sequence with ethyl acetate. All threeacetate-based solvents can be purchased in food grade quality. Thispresents an advantage when the extracted oligomeric fractions are to beused in a food or pharmaceutical product.

FIG. 1 depicts the high performance liquid chromatography (HPLC) traceof the cocoa polyphenol standard showing the individual monomers andoligomers.

FIG. 2 shows the selective extraction profile for methyl acetate. It hasbeen found that initial extraction of an aqueous cocoa polyphenolextract with methyl acetate will selectively extract catechin,epicatechin, and procyanidin dimers, trimers, tetramers, pentamers,hexamers, heptamers, octamers and minor amounts of procyanidin nonamers.It has also been found (not depicted) that further extraction withmethyl acetate, after the lower order oligomers have been removed, willselectively extract procyanidin nonamers and higher oligomers.

FIG. 3 shows the selective extraction profile for n-butyl acetate, whichpreviously was not known. It has been found that initial extraction ofan aqueous cocoa polyphenol extract with n-butyl acetate willselectively extract catechin, epicatechin and minor amounts ofprocyanidin dimers.

FIG. 4 shows the selective extraction profile for ethyl acetate. Aspreviously shown, initial extraction of an aqueous cocoa polyphenolextract with ethyl acetate will selectively extract catechin,epicatechin and procyanidin dimers, trimers and tetramers.

In addition to the three acetate-based solvents, solvents that are amixture of methyl acetate and diethyl ether may be used to selectivelyextract various fractions of procyanidin oligomers from the aqueouscocoa polyphenol extract. Such diethyl ether-based solvents may havemethyl acetate:diethyl ether ratios ranging from 0-100:100-0 (v/v).

As shown in FIG. 5, where the solvent has a methyl acetate:diethyl etherratio of 0:100 (v/v), that is, where the solvent is diethyl ether alone,the solvent selectively extracts catechin, epicatechin and procyanidindimers from the aqueous cocoa polyphenol extract. As shown in FIG. 6,where the solvent has a methyl acetate:diethyl ether ratio of 25:75(v/v), the solvent selectively extracts catechin, epicatechin andprocyanidin dimers, along with minor amounts of procyanidin trimers. Asshown in FIG. 7, where the mixed solvent has a methyl acetate:diethylether ratio of 50:50 (v/v), the solvent selectively extracts catechin,epicatechin and procyanidin dimers, trimers and tetramers. As shown inFIG. 8, where the mixed solvent has a methyl acetate:diethyl ether ratioof 75:25 (v/v), the solvent selectively extracts catechin, epicatechinand procyanidin dimers, trimers, tetramers, pentamers and minor amountsof procyanidin hexamers.

The table below summarizes the selection extraction profiles of theacetate- and diethyl ether-based organic solvents discussed herein.Solvent Extraction profile n-butyl acetate 1, 2(minor) Diethyl ether 1-225:75 methyl acetate:diethyl ether 1-2, 3(minor) Ethyl acetate 1-4 50:50methyl acetate:diethyl ether 1-4 75:25 methyl acetate:diethyl ether 1-5,6(minor) Methyl acetate 1-8, 9(minor); 9+

It is expected that other diethyl ether-based solvents, with otherratios of methyl acetate to diethyl ether, will selectively extractother groups of procyanidin oligomers.

There are two main factors in determining the solvents used in aparticular selective extraction sequence. The first factor is theextraction profiles for the individual solvents employed. As noted, theacetate solvents (n-butyl acetate, ethyl acetate, and methyl acetate)are limited to selectively extracting, respectively: catechin,epicatechin and minor amounts of procyanidin dimers; catechin,epicatechin and procyanidin dimers, trimers and tetramers; and catechin,epicatechin and procyanidin dimers, trimers, tetramers, pentamers,hexamers, heptamers, octamers and minor amounts of procyanidin nonamers.

By contrast, the diethyl ether-based solvent group have a greaterselectivity for procyanidin oligomers depending upon the proportion ofdiethyl ether in the solvent. For example, diethyl ether aloneselectively extracts catechin, epicatechin and procyanidin dimers. Amixture of methyl acetate and diethyl ether (25:75 v/v) selectivelyextracts catechin, epicatechin and procyanidin dimers and minor amountsof procyanidin trimers. And a mixture of methyl acetate and diethylether (75:25 v/v) selectively extracts catechin, epicatechin andprocyanidin dimers, trimers, tetramers, pentamers and minor amounts ofprocyanidin hexamers.

The second factor in determining the solvents to be used is the relativesolubility of the target procyanidin oligomeric fractions in the chosensolvent. Cocoa procyanidins tend to be more soluble in an acetate-basedsolvent as opposed to the combined methyl acetate:diethyl ether solventgenerating a similar extraction profile. For example ethyl acetate andan equal mixture of methyl acetate and diethyl ether (50:50 v/v) bothselectively extract catechin, epicatechin and procyanidin dimers,trimers and tetramers from an aqueous cocoa polyphenol extract. However,ethyl acetate requires fewer extraction washes to accumulate the targetpolyphenols. It is known that no solvent exhaustively extracts itstarget polyphenols in a single extraction wash. Multiple extractionwashes (as many as four) are required for the acetate solvents. Bycontrast, it is known that diethyl ether and diethyl ether-basedsolvents require further additional extraction washes (more than ten) toapproach exhaustion, and may never achieve exhaustive extraction.

Therefore, where available, substituting diethyl ether-based solventswith acetate-based solvents with corresponding extraction profilesimproves extraction efficiency.

Based on the selective extraction characteristics of the acetate- anddiethyl ether-based solvents, various selective extraction sequences maybe employed to selectively extract the desired procyanidin monomersand/or oligomer(s).

The aqueous cocoa polyphenol extract may be prepared by any conventionalmethod. For the selective extractions where the first solvent is n-butylacetate or methyl acetate, the aqueous cocoa polyphenol extract isprepared as follows:

Sample preparation: cocoa polyphenol extract. A cocoa polyphenol extractis prepared via a multi-step process aimed at minimizing loss of thecocoa procyanidins. Cacao seeds are harvested, washed free of pulp anddried. Under ambient conditions the dried beans are expeller-pressed toremove cocoa butter. The expeller cake is then ground and extracted withethanol:water (70:30 v/v). Solids are removed by centrifugation. Theliquid extract is evaporated under reduced pressure to remove theethanol and finally spray dried.

Sample preparation: cocoa polyphenol extract from cacao seeds. Freshunfermented cacao seeds (30 g) are freeze-dried (16.9 g). Freeze-driedseeds (10.2 g) are defatted with hexane, and a sub-sample of thedefatted beans (6.3 g) is milled and extracted thrice with 40 mLacetone:water:acetic acid (70:29.5:0.5, v/v/v) while sonicating (10 min,50° C.). Acetone is removed from the combined extractions by rotaryevaporation under reduced pressure. The remaining liquid is freeze driedto afford a red-purple residue (1.13 g).

The dried cocoa polyphenol extract is dissolved in water, preferably 50g of extract in 200 mL of water, to provide the aqueous cocoa polyphenolextract for selective extraction, beginning with n-butyl acetate ormethyl acetate.

Selectively extracted fractions were analyzed via HPLC using an Agilent1100 HPLC system equipped with an autosampler, quaternary HPLC pump,column heater, diode array detector, and fluorescence detector. Thecolumn used was Develosil Diol (250×4.6 mm I.D., 5 μ particle size)purchased from Phenomenex (Torrance, Calif.).

The binary mobile phase consisted of (A) acetonitrile:acetic acid (98:2,v/v) and (B) methanol:water:acetic acid (95:3:2, v/v/v). Separationswere effected by a linear gradient (of the mobile phase) at 35° C. withan 1.0 mL/min flow rate as follows: 0-35 minutes, 0% B; 35-40 min, 40% Bisocratic; 40-45 min, 40-0% B, followed by a 5 minute re-equilibratetime. Eluent was monitored by UV at 280 nm and fluorescence detection(FLD) (excitation wavelength=276 nm, emission wavelength=316 nm).Extracts and purified fractions were characterized by MS methods andparameters adapted from Hammerstone et al., J. Agric. and Food Chem. 47(1999) 490, which are incorporated herein by reference. Ionizationreagents were added via a tee in the eluant stream of the HPLC justprior to the mass spectrometer and delivered via an HPLC pump.Conditions for analysis in the positive ion mode included introductionof 0.05M NaCl at a flow rate of 0.05 mL/min to assist ionization, acapillary voltage of 3.5 kV, a fragmentor voltage of 100 V, a nebulizingpressure of 25 psig, and a drying gas temperature of 350° C. Conditionsfor analysis in the negative ion mode included 1.5 M ammonium hydroxideas a buffering agent at a flow rate of 0.09 mL/min for 29 minutes, andthen at 0.05 mL/min. Capillary voltage was 3 kV, fragmentor voltage was75 V, nebulizing pressure was 25 psig and drying gas temperature was350° C.

Use of ammonium hydroxide was omitted from the MS analysis. Samples weredissolved in acetone:water:acetic acid (70:29.5:0.5, v/v/v) or mobilephase and filtered through 0.45 μm PiFE syringe filters prior toinjection. All extractions were performed at room temperature. No acidswere used in the extraction solvents.

In light of the non-exhaustive nature of the extraction solvents usedherein, as used in the following examples, where the solvent is n-butylacetate, ethyl acetate or methyl acetate, “extracted” will mean up tofour extraction washes were performed with the solvent. When used inconjunction with diethyl ether or any mixture of diethyl ether andmethyl acetate, “extracted” will mean that at least ten extractionwashes were performed with the solvent. It will be understood that allorganic phases from all extraction runs are combined for use or furtherextraction, as desired.

Also as used herein, “enriched in” shall mean the following: where thesolvent is an acetate-based or diethyl ether-based organic solvent,“enriched in” shall mean that the identified phase consists essentiallyof the identified monomers and/or oligomers dissolved in the namedsolvent, together with trace amounts of other substances. Where thesolvent is water, that is, where the phase at issue is the aqueous phasefrom an extraction step, “enriched in” shall mean that the phaseconsists essentially of the identified monomers and/or oligomersdissolved in water, together with trace amounts of other substances.

It will be understood by those skilled in the art that the organic andaqueous phases resulting from each of the extractions in the followingexamples may be separated by conventional means and the separate phasesused as is, further extracted or discarded, as preferred. Wheredesirable, the solvent may be removed from an organic phase byconventional means and the resulting product dissolved in water toprovide an aqueous mixture. It will be further understood that where anorganic solvent phase (n-butyl acetate phase, ethyl acetate phase,methyl acetate phase, diethyl ether phase or other organic phase) isbeing extracted in an example, prior to extraction the solvent isremoved from the organic phase by evaporation or other conventionalmeans and the resulting product dissolved in water, providing an aqueoussolution of the product that may then be extracted.

The following examples may be more readily understood with reference toFIGS. 9-14. In the figures, “CP” refers to the aqueous extract of cocoapolyphenols, containing catechin, epicatechin and procyanidin dimers andhigher oligomers. “O” refers to the organic phase and “A” to the aqueousphase after extraction, as well as the phase (aqueous or organic) beingextracted. For the solvents, “nBA” refers to n-butyl acetate; “0:100”refers to a solvent containing only diethyl ether; “25:75” refers to adiethyl ether-based solvent where the ratio of methyl acetate to diethylether is 25:75 (v/v); “EtAc” refers to ethyl acetate; “75:25” refers toa diethyl ether-based solvent where the ratio of methyl acetate todiethyl ether is 75:25 (v/v); and “MeAc” refers to methyl acetate,without any diethyl ether. Numbers in parentheses refer to the degree(s)of polymerization of the oligomer(s) contained in an extracted phase.

In Examples 1 through 1.5.23, the initial extraction solvent is n-butylacetate.

The examples may be better understood with reference to the flowdiagrams in FIG. 9.

EXAMPLE 1

Initial extraction: n-butyl acetate: An aqueous mixture of cocoapolyphenols (catechin, epicatechin, and procyanidin dimers and higheroligomers) may be extracted with n-butyl acetate. The resulting n-butylphase is enriched in catechin and epicatechin, along with minor amountsof procyanidin dimers, and the resulting aqueous phase is enriched inprocyanidin dimers and higher oligomers. Compare FIGS. 1, 3.

EXAMPLE 1.1

Second extraction: diethyl ether: The aqueous phase enriched inprocyanidin dimers and higher oligomers (Example 1) may be extractedwith diethyl ether to provide an organic (diethyl ether) phase enrichedin procyanidin dimers and an aqueous phase enriched in procyanidintrimers and higher oligomers.

EXAMPLE 1.1.1

Third extraction: ethyl acetate: The aqueous phase enriched inprocyanidin trimers and higher oligomers (Example 1.1) may be extractedin ethyl acetate. The resulting ethyl acetate phase will be enriched inprocyanidin trimers and tetramers while the resulting aqueous phase isenriched in procyanidin pentamers and higher oligomers.

EXAMPLE 1.1.2

Fourth extraction: methyl acetate:diethyl ether (75:25 v/v)

The aqueous phase enriched in procyanidin pentamers and higher oligomers(Example 1.1.1) may be extracted with an organic solvent that is amixture of methyl acetate:diethyl ether (75:25 v/v) to provide anorganic phase enriched in procyanidin pentamers and an aqueous phaseenriched in procyanidin hexamers and higher oligomers.

EXAMPLE 1.1.3

Fifth extraction: methyl acetate: The aqueous phase enriched inprocyanidin hexamers and higher oligomers (Example 1.1.2) may beextracted with methyl acetate to provide a methyl acetate phase enrichedin procyanidin hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers and an aqueous phase enriched in procyanidinnonamers and higher oligomers.

EXAMPLE 1.1.4

Sixth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers (Example 1.1.3) may be extracted with methylacetate to provide an organic phase enriched in procyanidin nonamers andhigher oligomers. The resulting aqueous phase will be substantiallydepleted in procyanidins.

EXAMPLE 1.1.5

Fourth extraction: methyl acetate: In a variation on Example 1.1.2, thesolvent is removed from the organic phase of Example 1.1.1 byevaporation or other conventional means, and the resulting productdissolved in water. The dissolved product may be extracted with methylacetate to provide a methyl acetate phase enriched in procyanidinpentamers, hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers and an aqueous phase enriched in procyanidinnonamers and higher oligomers.

EXAMPLE 1.1.6

Fifth extraction: methyl acetate:diethyl ether (75:25 v/v): The solventis removed from the methyl acetate phase of Example 1.1.5 by evaporationor other conventional means, and the resulting product dissolved inwater. The dissolved product may be extracted with an organic solventthat is a mixture of methyl acetate and diethyl ether (75:25 v/v), toprovide an organic phase enriched in procyanidin pentamers, with minoramounts of procyanidin hexamers. The aqueous phase will be enriched inprocyanidin hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers.

EXAMPLE 1.1.7

Fifth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.1.5) may beextracted with methyl acetate to provide a methyl acetate phase enrichedin procyanidin nonamers and higher oligomers. The aqueous phase will besubstantially depleted in procyanidins.

EXAMPLE 1.1.8

Third extraction: methyl acetate:diethyl ether (75:25 v/v): In avariation on Example 1.1.1, the aqueous phase enriched in procyanidintrimers and higher oligomers (Example 1.1) may be extracted with anorganic solvent comprising a mixture of methyl acetate and diethyl ether(75:25 v/v). The resulting organic phase will be enriched in procyanidintrimers, tetramers and pentamers, with minor amounts of procyanidinhexamers. The aqueous phase will be enriched in procyanidin hexamers andhigher oligomers.

EXAMPLE 1.1.9

Fourth extraction: ethyl acetate: The solvent is removed from theorganic phase of Example 1.1.8 by evaporation or other conventionalmeans, and the resulting product dissolved in water. The dissolvedproduct may be extracted with ethyl acetate, providing an ethyl acetatephase enriched in procyanidin trimers and tetramers and a resultingaqueous phase enriched in procyanidin pentamers, with minor amounts ofprocyanidin hexamers.

EXAMPLE 1.1.10

Fourth extraction: methyl acetate: The aqueous phase enriched inprocyanidin hexamers and higher oligomers (Example 1.1.8) may beextracted with methyl acetate, providing a methyl acetate organic phaseenriched in procyanidin hexamers, heptamers, octamers and minor amountsof procyanidin nonamers, and an aqueous phase enriched in procyanidinnonamers and higher oligomers.

EXAMPLE 1.1.11

Fifth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.1.10) may beextracted with methyl acetate to provide a methyl acetate phase enrichedin procyanidin nonamers and higher oligomers. The aqueous phase issubstantially depleted of procyanidins.

EXAMPLE 1.1.12

Third extraction: methyl acetate: In another variation on Example 1.1.1,the aqueous phase enriched in procyanidin trimers and higher oligomers(Example 1.1) may be extracted with methyl acetate to provide a methylacetate phase enriched in procyanidin trimers, tetramers, pentamers,hexamers, heptamers, octamers and minor amounts of procyanidin nonamersand an aqueous phase enriched in procyanidin nonamers and higheroligomers.

EXAMPLE 1.1.13

Fourth extraction: ethyl acetate: The solvent is removed from the methylacetate phase of Example 1.1.12 by evaporation or other conventionalmeans, and the resulting product dissolved in water. The dissolvedproduct may be extracted with ethyl acetate to provide an ethyl acetatephase enriched in procyanidin trimers and tetramers. The aqueous phasewill be enriched in procyanidin pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers.

EXAMPLE 1.1.14

Fifth extraction: methyl acetate:diethyl ether (75:25 v/v): The aqueousphase enriched in procyanidin pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers (Example 1.1.13) may beextracted with an organic solvent that is a mixture of methyl acetateand diethyl ether (75:25 v/v) to provide an organic phase enriched inprocyanidin pentamers with minor amounts of procyanidin hexamers. Theaqueous phase will be enriched in procyanidin hexamers, heptamers,octamers and minor amounts of procyanidin nonamers.

EXAMPLE 1.1.15

Fourth extraction: methyl acetate:diethyl ether (75:25 v/v): In avariation on Example 1.1.13, the solvent is removed from the methylacetate phase of Example 1.1.12 by evaporation or other conventionalmeans, and the resulting product dissolved in water. The dissolvedproduct may be extracted with an organic solvent that is a mixture ofmethyl acetate and diethyl ether (75:25 v/v) to provide an organic phaseenriched in procyanidin trimers, tetramers and pentamers, with a minoramount of procyanidin hexamers. The aqueous phase will be enriched inprocyanidin hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers.

EXAMPLE 1.1.16

Fifth extraction: ethyl acetate: The solvent is removed from the organicphase of Example 1.1.15 by evaporation or other conventional means, andthe resulting product dissolved in water. The dissolved product may beextracted with ethyl acetate to provide an ethyl acetate phase enrichedin procyanidin trimers and tetramers and an aqueous phase enriched inprocyanidin pentamers, with minor amounts of procyanidin hexamers.

EXAMPLE 1.1.17

Fourth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.1.12) may beextracted with methyl acetate. The resulting methyl acetate phase willbe enriched in procyanidin nonamers and higher oligomers. The aqueousphase is substantially depleted of procyanidins.

EXAMPLE 1.2

Second extraction: methyl acetate:diethyl ether (25:75 v/v): In avariation on Example 1.1, the aqueous phase enriched in procyanidindimers and higher oligomers (Example 1) may be extracted with an organicsolvent that is a mixture of methyl acetate and diethyl ether (25:75v/v), to provide an organic phase enriched in procyanidin dimers, withminor amounts of procyanidin trimers, and an aqueous phase enriched inprocyanidin trimers and higher oligomers.

EXAMPLE 1.2.1

Third extraction: ethyl acetate: The aqueous phase enriched inprocyanidin trimers and higher oligomers (Example 1.2) may be extractedethyl acetate. The resulting ethyl acetate phase will be enriched inprocyanidin trimers and tetramers, while the aqueous phase will beenriched in procyanidin pentamers and higher oligomers.

EXAMPLE 1.2.2

Fourth extraction: methyl acetate:diethyl ether (75:25 v/v): The aqueousphase enriched in procyanidin pentamers and higher oligomers (Example1.2.1) may be extracted with an organic solvent comprising a mixture ofmethyl acetate and diethyl ether(75:25 v/v), to provide an organic phaseenriched in procyanidin pentamers and minor amounts of procyanidinhexamers. The aqueous phase will be enriched in procyanidin hexamers andhigher oligomers.

EXAMPLE 1.2.3

Fifth extraction: methyl acetate: The aqueous phase enriched inprocyanidin hexamers and higher oligomers (Example 1.2.2) may beextracted with methyl acetate to provide a methyl acetate phase enrichedin procyanidin hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers and an aqueous phase enriched in procyanidinnonamers and higher oligomers.

EXAMPLE 1.2.4

Sixth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.2.3) may beextracted in methyl acetate. The resulting methyl acetate phase will beenriched in procyanidin nonamers and higher oligomers, while the aqueousphase will be substantially depleted of procyanidins.

EXAMPLE 1.2.5

Fourth extraction: methyl acetate: In a variation of Example 1.2.2, theaqueous phase enriched in procyanidin pentamers and higher oligomers(Example 1.2.1) may be extracted with methyl acetate to provide a methylacetate phase enriched in procyanidin pentamers, hexamers, heptamers,octamers and minor amounts of procyanidin nonamers and an aqueous phaseenriched in procyanidin nonamers and higher oligomers.

EXAMPLE 1.2.6

Fifth extraction: methyl acetate:diethyl ether (75:25 v/v): The solventis removed from the organic phase of Example 1.2.5 by evaporation orother conventional means, and the resulting product dissolved in water.The dissolved product may be extracted with an organic solventcomprising a mixture of methyl acetate and diethyl ether (75:25 v/v).The resulting organic phase will be enriched in procyanidin pentamers,with minor amounts of procyanidin hexamers. The aqueous phase will beenriched in procyanidin hexamers, heptamers, octamers and minor amountsof procyanidin nonamers.

EXAMPLE 1.2.7

Fifth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.2.5) may beextracted with methyl acetate to provide a methyl acetate phase enrichedin procyanidin nonamers and higher oligomers. The aqueous phase will besubstantially depleted of procyanidins.

EXAMPLE 1.2.8

Third extraction: methyl acetate:diethyl ether (75:25 v/v): In avariation on Example 1.2.1, the aqueous phase enriched in procyanidintrimers and higher oligomers (Example 1.2) may be extracted with anorganic solvent comprising a mixture of methyl acetate and diethyl ether(75:25 v/v). The resulting organic phase will be enriched in procyanidintrimers, tetramers and pentamers, with minor amounts of procyanidinhexamers. The aqueous phase will be enriched in procyanidin hexamers andhigher oligomers.

EXAMPLE 1.2.9

Fourth extraction: methyl acetate: The aqueous phase procyanidinhexamers and higher oligomers (Example 1.2.8) may be extracted withmethyl acetate. The resulting methyl acetate phase will be enriched inprocyanidin hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers, and the aqueous phase will be enriched inprocyanidin nonamers and higher oligomers.

EXAMPLE 1.2.10

Fifth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.2.9) may beextracted with methyl acetate. The resulting methyl acetate phase willbe enriched in procyanidin nonamers and higher oligomers, while theaqueous phase will be substantially depleted of procyanidins.

EXAMPLE 1.2.11

Fourth extraction: ethyl acetate: The solvent is removed from theorganic phase of Example 1.2.8 by evaporation or other conventionalmeans, and the resulting product dissolved in water. The dissolvedproduct may be extracted with ethyl acetate. The resulting ethyl acetatephase will be enriched in procyanidin trimers and tetramers, while theaqueous phase will be enriched in procyanidin pentamers, with minoramounts of procyanidin hexamers.

EXAMPLE 1.2.12

Third extraction: methyl acetate: In another variation on Example 1.2.1,the aqueous phase enriched in procyanidin trimers and higher oligomers(Example 1.2) may be extracted with methyl acetate. The resulting methylacetate phase will be enriched in procyanidin trimers, tetramers,pentamers, hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers, while the aqueous phase will be enriched inprocyanidin nonamers and higher oligomers.

EXAMPLE 1.2.13

Fourth extraction: ethyl acetate: The solvent is removed from the methylacetate phase of Example 1.2.12 by evaporation or other conventionalmeans, and the resulting product dissolved in water. The dissolvedproduct may be extracted with ethyl acetate. The resulting ethyl acetatephase will be enriched in procyanidin trimers and tetramers, while theaqueous phase will be enriched in procyanidin pentamers, hexamers,heptamers, octamers and minor amounts of procyanidin nonamers.

EXAMPLE 1.2.14

Fifth extraction: methyl acetate:diethyl ether (75:25 v/v): The aqueousphase enriched in procyanidin pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers (Example 1.2.13) may beextracted with an organic solvent comprising a mixture of methyl acetateand diethyl ether (75:25 v/v). The resulting organic phase will beenriched in procyanidin pentamers, with minor amounts of procyanidinhexamers. The aqueous phase will be enriched in procyanidin hexamers,heptamers, octamers and minor amounts of procyanidin nonamers.

EXAMPLE 1.2.15

Fourth extraction: methyl acetate:diethyl ether (75:25 v/v): In avariation on Example 1.2.13, the solvent is removed from the methylacetate phase of Example 1.2.12 by evaporation or other conventionalmeans, and the resulting product dissolved in water. The dissolvedproduct may be extracted with an organic solvent comprising a mixture ofmethyl acetate and diethyl ether (75:25 v/v). The resulting organicphase will be enriched in proc yanidin trimers, tetramers and pentamers,with minor amounts of proc yani din hex amers. The aqueous phase will beenriched in procyanidin hexamers, heptamers, octamers and minor amountsof procyanidin nonamers.

EXAMPLE 1.2.16

Fifth extraction: ethyl acetate: The solvent is removed from the organicphase of Example 1.2.15 by evaporation or other conventional means, andthe resulting product dissolved in water. The dissolved product may beextracted with ethyl acetate. The resulting ethyl acetate phase will beenriched in procyanidin trimers and tetramers, while the aqueous phasewill be enriched in procyanidin pentamers, with minor amounts ofprocyanidin hexamers.

EXAMPLE 1.2.17

Fourth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.2.12) may beextracted with methyl acetate. The resulting methyl acetate phase willbe enriched in procyanidin nonamers and higher oligomers. The aqueousphase will be substantially depleted of procyanidins.

EXAMPLE 1.3

Second extraction: ethyl acetate: In a further variation on Example 1.1,the aqueous phase enriched in procyanidin dimers and higher oligomers(Example 1) may be extracted with ethyl acetate to provide an ethylacetate phase enriched in procyanidin dimers, trimers and tetramers, andan aqueous phase enriched in procyanidin pentamers and higher oligomers.

EXAMPLE 1.3.1

Third extraction: diethyl ether: The solvent is removed from the ethylacetate phase of Example 1.3 by evaporation or other conventional means,and the resulting product dissolved in water. The dissolved product maybe extracted with diethyl ether to provide a diethyl ether phaseenriched in procyanidin dimers and an aqueous phase enriched inprocyanidin trimers and tetramers.

EXAMPLE 1.3.2

Third extraction: methyl acetate:diethyl ether (25:75 v/v):Alternatively to Example 1.3.1, the solvent is removed from the ethylacetate phase of Example 1.3 by evaporation or other conventional means,and the resulting product dissolved in water. The dissolved product maybe extracted with an organic solvent comprising a mixture of methylacetate and diethyl ether (25:75 v/v), to provide an organic phaseenriched in procyanidin dimers with minor amounts of procyanidintrimers, and an aqueous phase enriched in procyanidin trimers andtetramers.

EXAMPLE 1.3.3

Third extraction: methyl acetate:diethyl ether (75:25 v/v): The aqueousphase enriched in procyanidin pentamers and higher oligomers (Example1.3) may be extracted with an organic solvent comprising a mixture ofmethyl acetate and diethyl ether (75:25 v/v), providing an organic phaseenriched in procyanidin pentamers and a minor amount of procyanidinhexamers, and a resulting aqueous phase enriched in procyanidin hexamersand higher oligomers.

EXAMPLE 1.3.4

Fourth extraction: methyl acetate: The aqueous phase enriched inprocyanidin hexamers and higher oligomers (Example 1.3.3) may beextracted with methyl acetate, providing a methyl acetate phase enrichedin procyanidin hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers, and an aqueous phase enriched in procyanidinnonamers and higher oligomers.

EXAMPLE 1.3.5

Fifth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.3.4) may beextracted with methyl acetate to provide a methyl acetate phase enrichedin procyanidin nonamers and higher oligomers. The aqueous phase will besubstantially depleted of procyanidins.

EXAMPLE 1.3.6

Third extraction: methyl acetate: In a variation on Example 1.3.3, theaqueous phase enriched in procyanidin pentamers and higher oligomers(Example 1.3) may be extracted with methyl acetate, to provide a methylacetate phase enriched in procyanidin pentamers, hexamers, heptamers,octamers and minor amounts of procyanidin nonamers, and an aqueous phaseenriched in procyanidin nonamers and higher oligomers.

EXAMPLE 1.3.7

Fourth extraction: methyl acetate:diethyl ether (75:25 v/v): The solventis removed from the methyl acetate phase of Example 1.3.6 by evaporationor other conventional means, and the resulting product dissolved inwater. The dissolved product may be extracted with an organic solventcomprising a mixture of methyl acetate and diethyl ether (75:25 v/v).The resulting organic phase will be enriched in procyanidin pentamerswith minor amounts of procyanidin hexamers. The aqueous phase will beenriched in procyanidin hexamers, heptamers, octamers and minor amountsof procyanidin nonamers.

EXAMPLE 1.3.8

Fourth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.3.6) may beextracted in methyl acetate, providing a methyl acetate phase enrichedin procyanidin nonamers and higher oligomers. The aqueous phase will besubstantially depleted of procyanidins.

EXAMPLE 1.4

Second extraction: methyl acetate:diethyl ether (75:25 v/v): In yet afurther variation on Example 1. 1, the aqueous phase enriched inprocyanidin dimers and higher oligomers (Example 1) may be extracted inan organic solvent comprising a mixture of methyl acetate and diethylether (75:25 v/v), to provide an organic phase enriched in procyanidindimers, trimers, tetramers and pentamers and a minor amount ofprocyanidin hexamers, and an aqueous phase enriched in procyanidinhexamers and higher oligomers.

EXAMPLE 1.4.1

Third extraction: diethyl ether: The solvent is removed from the organicphase of Example 1.4 by evaporation or other conventional means, and theresulting product dissolved in water. The dissolved product may beextracted with diethyl ether. The resulting diethyl ether phase will beenriched in procyanidin dimers and a resulting aqueous phase enriched inprocyanidin trimers, tetramers and pentamers, and a minor amount ofprocyanidin hexamers.

EXAMPLE 1.4.2

Fourth extraction: ethyl acetate: The aqueous phase enriched inprocyanidin trimers, tetramers and pentamers, and a minor amount ofprocyanidin hexamers (Example 1.4.1) may be extracted with ethylacetate, yielding an ethyl acetate phase enriched in procyanidin trimersand tetramers and a resulting aqueous phase enriched in procyanidinpentamers together with a minor amount of procyanidin hexamers.

EXAMPLE 1.4.3

Third extraction: methyl acetate:diethyl ether (25:75 v/v): In avariation on Example 1.4.1, the solvent is removed from the organicphase of Example 1.4 by evaporation or other conventional means, and theresulting product dissolved in water. The dissolved product may beextracted with an organic solvent comprising a mixture of methyl acetateand diethyl ether (25:75 v/v). The resulting organic phase will beenriched in procyanidin dimers and minor amounts of procyanidin trimers.The aqueous phase will be enriched in procyanidin trimers, tetramers,pentamers and minor amounts of procyanidin hexamers.

EXAMPLE 1.4.4

Fourth extraction: ethyl acetate: The aqueous phase procyanidin trimers,tetramers, pentamers and minor amounts of procyanidin hexamers (Example1.4.3) may be extracted with ethyl acetate to yield an ethyl acetatephase enriched in procyanidin trimers and tetramers an aqueous phaseenriched in procyanidin pentamers and minor amounts of procyanidinhexamers.

EXAMPLE 1.4.5

Third extraction: ethyl acetate: In another variation on Example 1.4.1,the solvent is removed from the organic phase of Example 1.4 byevaporation or other conventional means, the resulting product dissolvedin water, and the dissolved product extracted with ethyl acetate. Theresulting ethyl acetate phase will be enriched in procyanidin dimers,trimers and tetramers, while the aqueous phase will be enriched inprocyanidin pentamers and minor amounts of procyanidin hexamers.

EXAMPLE 1.4.6

Fourth extraction: diethyl ether: The solvent is removed from the ethylacetate phase of Example 1.4.5 by evaporation or other conventionalmeans, the resulting product dissolved in water, and the dissolvedproduct is extracted with diethyl ether. The resulting diethyl etherphase will be enriched in procyanidin dimers, while the aqueous phasewill be enriched in procyanidin trimers and tetramers.

EXAMPLE 1.4.7

Fourth extraction: methyl acetate:diethyl ether (25:75 v/v): In avariation on Example 1.4.6, the solvent is removed from the ethylacetate phase of Example 1.4.5 by evaporation or other conventionalmeans, the resulting product dissolved in water, and the dissolvedproduct is extracted with an organic solvent comprising a mixture ofmethyl acetate and diethyl ether (25:75 v/v). The resulting organicphase will be enriched in procyanidin dimers with a minor amount ofprocyanidin trimers. The aqueous phase will be enriched in procyanidintrimers and tetramers.

EXAMPLE 1.4.8

Third extraction: methyl acetate: The aqueous phase enriched inprocyanidin hexamers and higher oligomers (Example 1.4) may be extractedwith methyl acetate, yielding a methyl acetate phase enriched inprocyanidin hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers and an aqueous phase enriched in procyanidinnonamers and higher oligomers.

EXAMPLE 1.4.9

Fourth extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.4.8) may beextracted with methyl acetate, yielding a methyl acetate phase enrichedin procyanidin nonamers and higher oligomers. The aqueous phase issubstantially depleted of procyanidins.

EXAMPLE 1.5

Second extraction: methyl acetate: In yet another variation on Example1.1, the aqueous phase enriched in procyanidin dimers and higheroligomers (Example 1) may be extracted with methyl acetate, to provide amethyl acetate phase enriched in procyanidin dimers, trimers, tetramers,pentamers, hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers, and an aqueous phase enriched in procyanidinnonamers and higher oligomers.

EXAMPLE 1.5.1

Third extraction: diethyl ether: The solvent is removed from the methylacetate phase of Example 1.5 by evaporation or other conventional means,and the resulting product dissolved in water. The dissolved product maybe extracted with diethyl ether, to provide a diethyl ether phaseenriched in procyanidin dimers, and an aqueous phase enriched inprocyanidin trimers, tetramers, pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers.

EXAMPLE 1.5.2

Fourth extraction: ethyl acetate: The aqueous phase enriched inprocyanidin trimers, tetramers, pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers (Example 1.5.1) may beextracted with ethyl acetate, to provide an ethyl acetate phase enrichedin procyanidin trimers and tetramers, and an aqueous phase enriched inprocyanidin pentamers, hexamers, heptamers, octamers and minor amountsof procyanidin nonamers.

EXAMPLE 1.5.3

Fifth extraction: methyl acetate:diethyl ether (75:25 v/v): The aqueousphase enriched in procyanidin pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers (Example 1.5.2) may beextracted with an organic solvent comprising a mixture of methyl acetateand diethyl ether (75:25 v/v). The resulting organic phase will beenriched in procyanidin pentamers and minor amounts of procyanidinhexamers, while the aqueous phase will be enriched in procyanidinhexamers, heptamers, octamers and minor amounts of procyanidin nonamers.

EXAMPLE 1.5.4

Third extraction: ethyl acetate: In a variation on Example 1.5.1, thesolvent is removed from the methyl acetate phase of Example 1.5 byevaporation or other conventional means, the resulting product dissolvedin water, and the dissolved product is extracted with ethyl acetate. Theresulting ethyl acetate phase will be enriched in procyanidin dimers,trimers and tetramers, while the aqueous phase will be enriched inprocyanidin pentamers, hexamers, heptamers, octamers and minor amountsof procyanidin nonamers.

EXAMPLE 1.5.5

Fourth extraction: diethyl ether: The solvent is removed from the ethylacetate phase of Example 1.5.4 by evaporation or other conventionalmeans, the resulting product dissolved in water, and the dissolvedproduct is extracted with diethyl ether. The resulting diethyl etherphase will be enriched in procyanidin dimers, while the aqueous phasewill be enriched in procyanidin trimers and tetramers.

EXAMPLE 1.5.6

Fourth extraction: methyl acetate:diethyl ether (75:25 v/v): The aqueousphase enriched in procyanidin pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers (Example 1.5.4) may beextracted with an organic solvent comprising a mixture of methyl acetateand diethyl ether (75:25 v/v). The resulting organic phase will beenriched in procyanidin pentamers and minor amounts of procyanidinhexamers, while the aqueous phase will be enriched in procyanidinhexamers, heptamers, octamers and minor amounts of procyanidin nonamers.

EXAMPLE 1.5.7

Fourth extraction: methyl acetate:diethyl ether (75:25 v/v): The aqueousphase enriched procyanidin trimers, tetramers, pentamers, hexamers,heptamers, octamers and minor amounts of procyanidin nonamers (Example1.5.1) may be extracted with an organic solvent comprising a mixture ofmethyl acetate and diethyl ether (75:25 v/v). The resulting organicphase will be enriched in procyanidin trimers, tetramers and pentamersand minor amounts of procyanidin hexamers, while the aqueous phase willbe enriched in procyanidin hexamers, heptamers, octamers and minoramounts of procyanidin nonamers.

EXAMPLE 1.5.8

Fifth extraction: ethyl acetate: The solvent is removed from the organicphase of Example 1.5.7 by evaporation or other conventional means, theresulting product dissolved in water, and the dissolved product isextracted with ethyl acetate. The resulting ethyl acetate phase will beenriched in trimers and tetramers, while the aqueous phase will beenriched in procyanidin pentamers and minor amounts of procyanidinhexamers.

EXAMPLE 1.5.9

Fifth extraction: methyl acetate: The aqueous phase enriched inprocyanidin hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers (Example 1.5.7) may be extracted with methylacetate. The resulting methyl acetate phase will be enriched inprocyanidin hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers. The aqueous phase will be substantially depletedof procyanidins.

EXAMPLE 1.5.10

Third extraction: methyl acetate:diethyl ether (75:25 v/v): In anothervariation on Example 1.5.1, the solvent is removed from the methylacetate phase of Example 1.5 by evaporation or other conventional means,the resulting product dissolved in water, and the dissolved product isextracted with an organic solvent comprising a mixture of methyl acetateand diethyl ether (75:25 v/v). The resulting organic phase will beenriched in procyanidin dimers, trimers, tetramers and pentamers andminor amounts of procyanidin hexamers, while the aqueous phase will beenriched in procyanidin hexamers, heptamers, octamers and minor amountsof procyanidin nonamers.

EXAMPLE 1.5.11

Fourth extraction: diethyl ether: The solvent is removed from theorganic phase of Example 1.5.10 by evaporation or other conventionalmeans, the resulting product dissolved in water, and the dissolvedproduct is extracted with diethyl ether. The resulting diethyl etherphase will be enriched in procyanidin dimers while the aqueous phasewill be enriched in procyanidin trimers, tetramers, pentamers and minoramounts of procyanidin hexamers.

EXAMPLE 1.5.12

Fifth extraction: ethyl acetate: The aqueous phase enriched inprocyanidin trimers, tetramers pentamers and minor amounts ofprocyanidin hexamers (Example 1.5.11) may be extracted with ethylacetate. The resulting ethyl acetate phase will be enriched inprocyanidin trimers and tetramers, while the aqueous phase will beenriched in procyanidin pentamers and minor amounts of procyanidinhexamers.

EXAMPLE 1.5.13

Fourth extraction: ethyl acetate: In a variation on Example 1.5.11, thesolvent is removed from the organic phase of Example 1.5.10 byevaporation or other conventional means, the resulting product dissolvedin water, and the dissolved product is extracted with ethyl acetate. Theresulting ethyl acetate phase will be enriched in procyanidin dimers,trimers and tetramers, while the aqueous phase will be enriched inprocyanidin pentamers and minor amounts of procyanidin hexamers.

EXAMPLE 1.5.14

Fifth extraction: diethyl ether: The solvent is removed from the ethylacetate phase of Example 1.5.13 by evaporation or other conventionalmeans, the resulting product dissolved in water, and the dissolvedproduct is extracted with diethyl ether. The resulting diethyl etherphase will be enriched in procyanidin dimers, while the aqueous phasewill be enriched in procyanidin trimers and tetramers.

EXAMPLE 1.5.15

Fifth extraction: methyl acetate:diethyl ether (25:75 v/v): In avariation on Example 1.5.14, the solvent is removed from the ethylacetate phase of Example 1.5.13 by evaporation or other conventionalmeans, the resulting product dissolved in water, and the dissolvedproduct is extracted with an organic solvent comprising a mixture ofmethyl acetate and diethyl ether (25:75 v/v). The resulting organicphase will be enriched in dimers and minor amounts of procyanidintrimers, and the aqueous phase will be enriched in procyanidin trimersand tetramers.

EXAMPLE 1.5.16

Fourth extraction: methyl acetate:diethyl ether (25:75 v/v): In anothervariation on Example 1.5.11, the solvent is removed from the organicphase of Example 1.5.10 by evaporation or other conventional means, theresulting product dissolved in water, and the dissolved product isextracted with an organic solvent comprising a mixture of methyl acetateand diethyl ether (25:75 v/v). The resulting organic phase will beenriched in procyanidin dimers and minor amounts of procyanidin trimers.The aqueous phase will be enriched in procyanidin trimers, tetramers andpentamers and minor amounts of procyanidin hexamers.

EXAMPLE 1.5.17

Fifth extraction: ethyl acetate: The aqueous phase enriched inprocyanidin trimers, tetramers, pentamers and minor amounts ofprocyanidin hexamers (Example 1.5.16) may be extracted with ethylacetate. The resulting ethyl acetate phase will be enriched inprocyanidin trimers and tetramers, while the aqueous phase will beenriched in procyanidin pentamers and minor amounts of procyanidinhexamers.

EXAMPLE 1.5.18

Third extraction: methyl acetate:diethyl ether (25:75 v/v): In yetanother variation on Example 1.5.1, the solvent is removed from themethyl acetate phase of Example 1.5 by evaporation or other conventionalmeans, the resulting product dissolved in water, and the dissolvedproduct is extracted with an organic solvent comprising a mixture ofmethyl acetate and diethyl ether (25:75 v/v). The resulting organicphase will be enriched in procyanidin dimers and minor amounts ofprocyanidin trimers, while the aqueous phase will be enriched inprocyanidin trimers, tetramers, pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers.

EXAMPLE 1.5.19

Fourth extraction: ethyl acetate: The aqueous phase enriched inprocyanidin trimers, tetramers, pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers (Example 1.5.18) may beextracted with ethyl acetate. The resulting ethyl acetate phase will beenriched in procyanidin trimers and tetramers, while the aqueous phasewill be enriched in procyanidin pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers.

EXAMPLE 1.5.20

Fifth extraction: methyl acetate:diethyl ether (75:25 v/v): The aqueousphase enriched in procyanidin pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers (Example 1.5.19) may beextracted with an organic solvent comprising a mixture of methyl acetateand diethyl ether (75:25 v/v). The resulting organic phase will beenriched in procyanidin pentamers and minor amounts of procyanidinhexamers, while the aqueous phase will be enriched in procyanidinhexamers, heptamers, octamers and minor amounts of procyanidin nonamers.

EXAMPLE 1.5.21

Fourth extraction: methyl acetate:diethyl ether (75:25 v/v): In avariation on Example 1.5.19, the aqueous phase enriched in procyanidintrimers, tetramers, pentamers, hexamers, heptamers, octamers and minoramounts of procyanidin nonamers (Example 1.5.18) may be extracted withan organic solvent comprising a mixture of methyl acetate and diethylether (75:25 v/v). The resulting organic phase will be enriched inprocyanidin trimers, tetramers, pentamers and minor amounts ofprocyanidin hexamers, while the aqueous phase will be enriched inprocyanidin hexamers, heptamers, octamers and minor amounts ofprocyanidin nonamers.

EXAMPLE 1.5.22

Fifth extraction: ethyl acetate: The solvent is removed from the organicphase of Example 1.5.21 by evaporation or other conventional means, theresulting product dissolved in water, and the dissolved product isextracted with ethyl acetate. The resulting ethyl acetate phase will beenriched in procyanidin trimers and tetramers, while the aqueous phasewill be enriched in procyanidin pentamers and minor amounts ofprocyanidin hexamers.

EXAMPLE 1.5.23

Third extraction: methyl acetate: The aqueous phase enriched inprocyanidin nonamers and higher oligomers (Example 1.5) may be extractedwith methyl acetate to provide a methyl acetate phase enriched inprocyanidin nonamers and higher oligomers. The aqueous phase will besubstantially depleted in procyanidins.

Examples of other extraction sequences, using other initial extractionsolvents, are described in FIGS. 10-14. FIG. 10 depicts Examples 2through 2.5.2, wherein the initial extraction solvent (Example 2) isethyl acetate. FIG. 11 depicts Examples 3 through 3.6, wherein theinitial extraction solvent (Example 3) is methyl acetate. FIG. 12depicts Examples 4 through 4.4.5, wherein the initial extraction solventis diethyl ether. FIG. 13 depicts Examples 5 through 5.4.5, wherein theinitial extraction solvent is a mixture of methyl acetate and diethylether (25:75 v/v). FIG. 14 depicts Examples 6 through 6.5.1, wherein theinitial extraction solvent is a mixture of methyl acetate and diethylether (75:25 v/v).

It will be understood by those skilled in the art that, while the aboveexamples relate to selective extractions of aqueous cocoa polyphenolextracts, any substance comprising polar protic oligomers may beselectively extracted in accordance with the provided example. Suitablepolar protic oligomers include proanthocyanidins, hydrolyzable tannins,oligosaccharides, oligonucleotides, peptides, acrylamides, polysorbates,polyketides, poloxamers, polyethylene glycols, polyoxyethylene alcoholsand polyvinyl alcohols. Where the polar protic oligomers areproanthocyanidins, they may be proapigeninidins, proluteolinidins,protricetinidins, propelargonidins, prodelphinidins, proguibourtinidins,profisetinidins, prorobinetindins, proteracacinidins and/orpromelacacinidins.

While the invention has been described with respect to certain specificembodiments, it will be appreciated that many modifications and changesmay be made by those skilled in the art without departing from theinvention. It is intended, therefore, by the appended to cover all suchmodifications and changes as may fall within the true spirit and scopeof the invention.

1. A process for selectively extracting cocoa procyanidins from anaqueous mixture of cocoa polyphenols, which process comprises the stepof extracting the aqueous mixture with n-butyl acetate and separating anaqueous phase and an n-butyl acetate phase; whereby the aqueous phase isenriched in procyanidin dimers and higher oligomers and the n-butylacetate phase is enriched in catechin and epicatechin.
 2. The process ofclaim 1, further comprising the step of extracting the aqueous phasewith an organic solvent selected from the group consisting of diethylether and a mixture of methyl acetate and diethyl ether effective toselectively extract catechin, epicatechin and procyanidin dimers andseparating an aqueous phase and an organic phase; whereby the aqueousphase is enriched in procyanidin trimers and higher oligomers and theorganic phase is enriched in procyanidin dimers.
 3. The process of claim2, further comprising the step of extracting the aqueous phase enrichedin procyanidin trimers and higher oligomers with ethyl acetate andseparating an aqueous phase and an ethyl acetate phase; whereby theaqueous phase is enriched in procyanidin pentamers and higher oligomersand the ethyl acetate phase is enriched in procyanidin trimers andtetramers.
 4. The process of claim 3, further comprising the step ofextracting the aqueous phase enriched in procyanidin pentamers andhigher oligomers with an organic solvent which is a mixture of methylacetate and diethyl ether effective to selectively extract catechin,epicatechin and procyanidin dimers, trimers, tetramers and pentamers andseparating an aqueous phase and an organic phase; whereby the aqueousphase is enriched in procyanidin hexamers and higher oligomers and theorganic phase is enriched in procyanidin pentamers.
 5. The process ofclaim 4, further comprising the step of extracting the aqueous phaseenriched in procyanidin hexamers and higher oligomers with methylacetate and separating an aqueous phase and a methyl acetate phase;whereby the aqueous phase is enriched in procyanidin nonamers and higheroligomers and the methyl acetate phase is enriched in procyanidinhexamers, heptamers and octamers.
 6. The process of claim 5, furthercomprising the step of extracting the aqueous phase enriched inprocyanidin nonamers and higher oligomers with methyl acetate andseparating an aqueous phase and a methyl acetate phase enriched inprocyanidin nonamers and higher oligomers.
 7. The process of claim 2,further comprising the step of extracting the aqueous phase enriched inprocyanidin trimers and higher oligomers with methyl acetate andseparating an aqueous phase and a methyl acetate phase; whereby theaqueous phase is enriched in procyanidin nonamers and higher oligomersand the methyl acetate phase is enriched in procyanidin trimers,tetramers, pentamers, hexamers, heptamers and octamers.
 8. The processof claim 7, further comprising the step of removing the solvent from themethyl acetate phase, dissolving a product obtained thereby in water,extracting the dissolved product with ethyl acetate, and separating anaqueous phase and an ethyl acetate phase; whereby the aqueous phase isenriched in procyanidin pentamers, hexamers, heptamers and octamers andthe ethyl acetate phase is enriched in procyanidin trimers andtetramers.
 9. The process of claim 8, further comprising the step ofextracting the aqueous phase enriched in procyanidin pentamers,hexamers, heptamers and octamers with an organic solvent which is amixture of methyl acetate and diethyl ether effective to selectivelyextract catechin, epicatechin and procyanidin dimers, trimers, tetramersand pentamers and separating an aqueous phase and an organic phase;whereby the aqueous phase is enriched in procyanidin hexamers, heptamersand octamers and the organic phase is enriched in procyanidin pentamers.10. The process of claim 7, further comprising the step of extractingthe aqueous phase enriched in procyanidin nonamers and higher oligomerswith methyl acetate and separating an aqueous phase and a methyl acetatephase; whereby the methyl acetate phase is enriched in procyanidinnonamers and higher oligomers.
 11. The process of claim 1, furthercomprising the step of extracting the aqueous phase with ethyl acetateand separating an aqueous phase and an ethyl acetate phase; whereby theaqueous phase is enriched in procyanidin pentamers and higher oligomersand the ethyl acetate phase is enriched in procyanidin dimers, trimersand tetramers.
 12. The process of claim 11, further comprising the stepof extracting the aqueous phase enriched in procyanidin pentamers andhigher oligomers with an organic solvent which is a mixture of methylacetate and diethyl ether effective to selectively extract catechin,epicatechin and procyanidin dimers, trimers, tetramers and pentamers andseparating an aqueous phase and an organic phase; whereby the aqueousphase is enriched in procyanidin hexamers and higher oligomers and theorganic phase is enriched in procyanidin pentamers.
 13. The process ofclaim 12, further comprising the step of extracting the aqueous phaseenriched in procyanidin hexamers and higher oligomers with methylacetate and separating an aqueous phase and a methyl acetate phase;whereby the aqueous phase is enriched in procyanidin nonamers and higheroligomers and wherein the methyl acetate phase is enriched inprocyanidin hexamers, heptamers and octamers.
 14. The process of claim13, further comprising the step of extracting the aqueous phase enrichedin procyanidin nonamers and higher oligomers with methyl acetate andseparating an aqueous phase and a methyl acetate phase; whereby themethyl acetate phase is enriched in procyanidin nonamers and higheroligomers.
 15. The process of claim 11, further comprising the step ofextracting the aqueous phase enriched in procyanidin pentamers andhigher oligomers with methyl acetate and separating an aqueous phase anda methyl acetate phase; whereby the aqueous phase is enriched inprocyanidin nonamers and higher oligomers and the methyl acetate phaseis enriched in procyanidin pentamers, hexamers, heptamers and octamers.16. The process of claim 15, further comprising the step of removing thesolvent from the methyl acetate phase, dissolving a product obtainedthereby in water, extracting the dissolved product with an organicsolvent which is a mixture of methyl acetate and diethyl ether effectiveto selectively extract catechin, epicatechin and procyanidin dimers,trimers, tetramers and pentamers, and separating an aqueous phase and anorganic phase; whereby the aqueous phase is enriched in procyanidinhexamers, heptamers and octamers and the organic phase is enriched inprocyanidin pentamers.
 17. The process of claim 11, further comprisingthe step of removing the solvent from the ethyl acetate phase,dissolving a product obtained thereby in water and extracting thedissolved product in an organic solvent selected from the groupconsisting of diethyl ether and a mixture of methyl acetate and diethylether effective to selectively extract catechin, epicatechin andprocyanidin dimers, and separating an aqueous phase and an organicphase; whereby the aqueous phase is enriched in procyanidin trimers andtetramers and the organic phase is enriched in procyanidin dimers. 18.The process of claim 1, further comprising the step of extracting theaqueous phase with an organic solvent which is a mixture of methylacetate and diethyl ether effective to selectively extract catechin,epicatechin and procyanidin dimers, trimers, tetramers and pentamers andseparating an aqueous phase and an organic phase; whereby the aqueousphase is enriched in procyanidin hexamers and higher oligomers and theorganic phase is enriched in procyanidin dimers, trimers, tetramers andpentamers.
 19. The process of claim 18, further comprising the step ofextracting the aqueous phase enriched in procyanidin hexamers and higheroligomers with methyl acetate and separating an aqueous phase and amethyl acetate phase; whereby the aqueous phase is enriched inprocyanidin nonamers and higher oligomers and the methyl acetate phaseis enriched in procyanidin hexamers, heptamers and octamers.
 20. Theprocess of claim 19, further comprising the step of extracting theaqueous phase enriched in procyanidin nonamers and higher oligomers withmethyl acetate and separating an aqueous phase and a methyl acetatephase; whereby the methyl acetate phase is enriched in procyanidinnonamers and higher oligomers.
 21. The process of claim 18, furthercomprising the step of removing the solvent from the organic phase,dissolving a product obtained thereby in water, extracting the dissolvedproduct with an organic solvent selected from the group consisting ofdiethyl ether and a mixture of methyl acetate and diethyl ethereffective to selectively extract catechin, epicatechin and procyanidindimers and separating an aqueous phase and an organic phase; whereby theaqueous phase is enriched in procyanidin trimers, tetramers andpentamers and the organic phase is enriched in procyanidin dimers. 22.The process of claim 21, further comprising the step of extracting theaqueous phase enriched in procyanidin trimers, tetramers and pentamerswith ethyl acetate and separating an aqueous phase and an ethyl acetatephase; whereby the aqueous phase is enriched in procyanidin pentamersand the ethyl acetate phase is enriched in procyanidin trimers andtetramers.
 23. The process of claim 1, further comprising the step ofextracting the aqueous phase with methyl acetate and separating anaqueous phase and a methyl acetate phase; whereby the aqueous phase isenriched in procyanidin nonamers and higher oligomers and the methylacetate phase is enriched in procyanidin dimers, trimers, tetramers,pentamers, hexamers, heptamers and octamers.
 24. The process of claim23, further comprising the step of removing the solvent from the methylacetate phase, dissolving a product obtained thereby in water,extracting the dissolved product with an organic solvent selected fromthe group consisting of diethyl ether and a mixture of methyl acetateand diethyl ether effective to selectively extract catechin, epicatechinand procyanidin dimers, and separating an aqueous phase and an organicphase; whereby the aqueous phase is enriched in procyanidin trimers,tetramers, pentamers, hexamers, heptamers and octamers and the organicphase is enriched in procyanidin dimers.
 25. The process of claim 24,further comprising the step of extracting the aqueous phase enriched inprocyanidin trimers, tetramers, pentamers, hexamers, heptamers, octamersand minor amounts of procyanidin nonamers with ethyl acetate andseparating an aqueous phase and an ethyl acetate phase; whereby theaqueous phase is enriched in procyanidin pentamers, hexamers, heptamers,octamers and minor amounts of procyanidin nonamers and the ethyl acetatephase is enriched in procyanidin trimers and tetramers.
 26. The processof claim 25, further comprising the step of extracting the aqueous phaseenriched in procyanidin pentamers, hexamers, heptamers and octamers withan organic solvent which is a mixture of methyl acetate and diethylether effective to selectively extract catechin, epicatechin andprocyanidin dimers, trimers, tetramers and pentamers and separating anaqueous phase and an organic phase; whereby the aqueous phase isenriched in procyanidin hexamers, heptamers and octamers and the organicphase is enriched in procyanidin pentamers.
 27. The process of claim 23,further comprising the step of removing the solvent from the methylacetate phase, dissolving a product obtained thereby in water,extracting the dissolved product with an organic solvent which is amixture of methyl acetate and diethyl ether effective to selectivelyextract catechin, epicatechin and procyanidin dimers, trimers, tetramersand pentamers and separating an aqueous phase and an organic phase;whereby the aqueous phase is enriched in procyanidin hexamers, heptamersand octamers and the organic phase is enriched in procyanidin dimers,trimers, tetramers and pentamers.
 28. The process of claim 27, furthercomprising the step of removing the solvent from the organic phase,dissolving a product obtained thereby in water, extracting the dissolvedproduct with an organic solvent selected from the group consisting ofdiethyl ether and a mixture of methyl acetate and diethyl ethereffective to selectively extract catechin, epicatechin and procyanidindimers and separating an aqueous phase and an organic phase; wherein theaqueous phase is enriched in procyanidin trimers, tetramers andpentamers and the organic phase is enriched in procyanidin dimers. 29.The process of claim 28, further comprising the step of extracting theaqueous phase enriched in procyanidin trimers, tetramers and pentamerswith ethyl acetate and separating an aqueous phase and an ethyl acetatephase; whereby the aqueous phase is enriched in procyanidin pentamersand the ethyl acetate phase is enriched in procyanidin trimers andtetramers.
 30. The process of claims 2, 17, 21, 24 or 28, wherein themixture of methyl acetate and diethyl ether is approximately 25:75(v/v).
 31. The process of claims 4, 9, 12, 16, 18, 26 or 27, wherein themixture of methyl acetate and diethyl ether is approximately 75:25(v/v).